Ang-(1-7) derviative oligopeptides and methods for using and producing the same

ABSTRACT

The present invention provides oligopeptides, in particular, Ang-(1-7) derivatives, and methods for using and producing the same. In one particular embodiment, oligopeptides of the invention have higher blood-brain barrier penetration and/or in vivo half-life compared to the native Ang-(1-7), thereby allowing oligopeptides of the invention to be used in a wide variety of clinical applications including in treatment of cognitive dysfunction and/of impairment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 62/027,219, filed Jul. 21, 2014, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to oligopeptides, such as Ang-(1-7)derivatives, and methods for using and producing the same. In oneparticular embodiment, oligopeptides of the invention have higherblood-brain barrier penetration and/or longer in vivo half-life comparedto the native Ang-(1-7), thereby allowing compounds of the invention tobe used in a wide variety of clinical applications to treat cognitivedysfunction and/or impairment.

BACKGROUND OF THE INVENTION

Cognitive dysfunction or impairment is a common neurologicalcomplication of congestive heart failure (“CHF”) and post cardiacsurgery affecting approximately 50-70% of patients at hospital dischargeand 20-40% of patients six months after surgery. The occurrence of CHFand postoperative cognitive dysfunction is associated with increasedduration of hospitalization and impaired long-term quality of life.Without being bound by any theory, it is believed that in general anyclinical condition associated with an increase in inflammatory cytokinesand/or increase in reactive oxygen species in central nervous system, inparticular in the brain, can lead to cognitive dysfunction.

Unfortunately, currently there is no effective pharmacological treatmentfor cognitive impairment or dysfunction for CHF and postoperativepatients or for any other clinical condition associated with an increasein inflammation cytokines and/or increase in reactive oxygen species inthe brain.

The present inventors have shown that CHF results in a significantimpairment of both spatial memory and object recognition ability. Thepresent inventors have also discovered that systemic administration ofnative Ang-(1-7) attenuates CHF-induced spatial memory and objectrecognition impairment. In addition, Mas, the receptor for Ang-(1-7), isknown to be expressed in the hippocampus. In addition, other researchersusing two different rat models have shown that Ang-(1-7) protects thecortex against reactive oxygen species (“ROS”)-mediated damage fromcerebral ischemia. This strongly implicates that the neuroprotectiveability of Ang-(1-7) against CHF-induced cognitive impairment ismediated by central activation of the Ang-(1-7)/Mas signaling axis atboth the vascular endothelial and neuronal levels.

Unfortunately, it is generally well known that oligopeptides, such asAng-(1-7) are relatively easily degraded in vivo and/or are not suitablefor conventional administration as Ang-(1-7) cannot readily cross theblood-brain barrier.

Accordingly, there is a need for Ang-(1-7) derivatives that canrelatively readily cross the blood-brain barrier and/or have asubstantially longer in vivo half-life compared to the native Ang-(1-7).

SUMMARY OF THE INVENTION

Some aspects of the invention provide an oligopeptide that isangiotensin-(1-7), i.e., “Ang-(1-7)”, derivative. Oligopeptides of theinvention have a longer in vivo half-life and/or increased blood-brainbarrier penetration than Ang-(1-7). In some embodiments, theoligopeptides of the invention have seven or eight amino acids.

One particular aspect of the invention provides an oligopeptidederivative of the formula: A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸ (SEQ ID NO:1), whereA¹ is selected from the group consisting of aspartic acid, glutamicacid, alanine, and a derivative thereof; A² is selected from the groupconsisting of arginine, histidine, lysine, and a derivative thereof; A³is selected from the group consisting of valine, alanine, isoleucine,leucine, and a derivative thereof; A⁴ is selected from the groupconsisting of tyrosine, phenylalanine, tryptophan, and a derivativethereof; A⁵ is selected from the group consisting of isoleucine, valine,alanine, leucine, and a derivative thereof; A⁶ is selected from thegroup consisting of histidine, arginine, lysine, and a derivativethereof; A⁷ is selected from the group consisting of proline, glycine,serine, and a derivative thereof; and A⁸ can be present or absent,wherein when A⁸ is present, A⁸ is selected from the group consisting ofserine, threonine, hydroxyproline, and a derivative thereof, provided(i) at least one of A¹-A⁸ is optionally substituted with a mono- ordi-carbohydrate; or (ii) when A⁸ is absent: (a) at least one of A¹-A⁷ issubstituted with a mono- or di-carbohydrate, (b) A⁷ is terminated withan amino group, or (c) a combination thereof.

In some embodiments, carbohydrate comprises glucose, galactose, xylose,fucose, rhamnose, lactose, cellobiose, melibiose, or a combinationthereof. In another embodiment, A⁸ is serine or a derivative thereof.

Still in other embodiments, (i) A⁸ is terminated with an amino group; or(ii) when A⁸ is absent, A⁷ is terminated with an amino group. Withinthese embodiments, in some instances (i) A⁸ is serine that isglycosylated with glucose or lactose; or (ii) when A⁸ is absent, A⁷ isserine that is glycosylated with glucose or lactose. Still in otherinstances, when A⁸ is absent and A⁷ serine that is glycosylated withglucose. Within the latter instances, in some cases A⁷ is terminatedwith an amino group.

Yet in other embodiments, A¹ is aspartic acid; A² is arginine; A³ isvaline; A⁴ is tyrosine; A⁵ is isoleucine; A⁶ is histidine; and (i) A⁸ isabsent and A⁷ is terminated with an amino group or A⁷ is a glycosylatedserine, or (ii) A⁸ is serine terminated with an amino group. Withinthese embodiments, in some cases A⁸ is a glycosylated serine. Still inother cases, A⁸ is absent and A⁷ is a glycosylated serine that isterminated with an amino group.

Another aspect of the invention provides a glycosylated Ang-(1-7)derivative having eight amino acids or less, typically seven or eightamino acids (e.g., amino acid residues). In some embodiments, theglycosylated Ang-(1-7) derivative is glycosylated with xylose, fucose,rhamnose, glucose, lactose, cellobiose, melibiose, or a combinationthereof. Still in other embodiments, the carboxylic acid end of saidglycosylated Ang-(1-7) derivative is substituted with an amino group.

Other aspects of the invention provide methods for treating cognitivedysfunction and/or impairment in a subject by administering atherapeutically effective amount of an oligonucleotide of the invention.In general, oligopeptides of the invention can be used to treat anyclinical condition that can be treated with Ang-(1-7).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing some of the oligopeptides of the invention andnative Ang-(1-7) to activate human umbilical vascular endothelial cells(HUVEC) in culture.

FIG. 2 is a graph showing NO production assay results for nativeAng-(1-7) and oligopeptides PN-A3, PN-A4 and PN-A5 of the invention.

FIG. 3A is a graph showing the select Mas receptor antagonists A779blocks NO production induced by oligopeptide PN-A5 of the invention.

FIG. 3B is a graph showing the averaged effect of the select Masreceptor antagonists A779 on NO production induced by oligopeptidePN-A5.

FIG. 4 is a graph showing the effects of oligopeptide PN-A5 on heartfailure induced object recognition memory impairment.

FIG. 5 is a graph showing the effects of oligopeptide PN-A5 on heartfailure induced spatial memory impairment.

FIG. 6A is a graph showing oligopeptide PN-A5 attenuates CIBP acutely.

FIG. 6B is a graph showing the results of tactile allodynia test usingvon Frey filaments.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “native” refers to any sequence of L amino acids used as astarting sequence or a reference for the preparation of partial orcomplete retro, inverso or retro-inverso analogues. Thus, the term“native Ang-(1-7)” refers to an oligopeptide having the same amino acidsequence as that of endogenous Ang-(1-7). It should be appreciated thatthe use of the term “native” does NOT imply naturally occurring,although it can include naturally occurring Ang-(1-7). The term “native”merely refers to having the same amino acid sequence as that ofAng-(1-7) without any modification of the amino acid residues.Accordingly, the term “native Ang-(1-7)” includes both syntheticAng-(1-7) and naturally occurring Ang-(1-7) as long as the amino acidresidues are the same and are not modified.

The term “Ang-(1-7) derivative” refers to oligopeptide in which one ormore amino acid residue is either modified or different than the aminoacid residue of the corresponding native Ang-(1-7). The term “Ang-(1-7)derivative” also includes oligopeptide of eight amino acid residues asdiscussed in more detail below.

The term “retro modified” refers to a peptide which is made up ofL-amino acids in which the amino acid residues are assembled in oppositedirection to the native peptide with respect the which it is retromodified. The term “inverso modified” refers to a peptide which is madeup of D-amino acids in which the amino acid residues are assembled inthe same direction as the native peptide with respect to which it isinverso modified. The term “retro-inverso modified” refers to a peptidewhich is made up of D-amino acids in which the amino acid residues areassembled in the opposite direction to the native peptide with respectto which it is retro-inverso modified. Thus, native Ang-(1-7) (L-aminoacids, N→C direction) is: Asp-Arg-Val-Tyr-Ile-His-Pro, i.e., DRVYIHP(SEQ ID NO:2). Retro-inverso Ang-(1-7) (D-amino acids, C→N direction)is: DRVYIHP (SEQ ID NO:3). Retro Ang-(1-7) (L-amino acids, C→Ndirection) is: DRVYIHP (SEQ ID NO:4). And inverso Ang-(1-7) (D-aminoacids, N→C direction) is: DRVYIHP (SEQ ID NO:5). The use of D-aminoacids in the context of inverso modified and retro-inverso modifiedAng-(1-7) derivatives is not intended to be limiting on the use ofD-amino amino acids in the oligopeptides. As discussed in more detailbelow, fewer than all of the amino acids in an Ang-(1-7) derivative maybe D-amino acids.

The term “carbohydrate” refers to pentose and hexose of empiricalformula (CH₂O)_(n), where n is 5 for pentose and 6 for hexose. Acarbohydrate can be monosaccharide, disaccharide, oligosaccharide (e.g.,3-20, typically 3-10, and often 3-5 monomeric saccharides are linkedtogether), or polysaccharide (e.g., greater than 20 monomeric saccharideunits). More often, the term carbohydrate refers to monosaccharideand/or disaccharide. However, it should be appreciated that the scope ofthe invention is not limited to mono- or di-saccharides. Often the terms“carbohydrate” and “saccharide” are used interchangeably herein.

The term “oligopeptide” as used throughout the specification and claimsis to be understood to include amino acid chain of any length, buttypically amino acid chain of about fifteen or less, often ten or less,still more often eight or less, and most often seven or eight.

It should be appreciated that one or more of the amino acids ofAng-(1-7) can be replaced with an “equivalent amino acid”, for example,L (leucine) can be replaced with isoleucine or other hydrophobicside-chain amino acid such as alanine, valine, methionine, etc., andamino acids with polar uncharged side chain can be replaced with otherpolar uncharged side chain amino acids. While Ang-(1-7) comprises 7amino acids, in some embodiments the oligopeptide of the invention haseight or less amino acids.

The term “derivative” refers to any chemical modification of the aminoacid, such as alkylation (e.g., methylation or ethylation) of the aminogroup or the functional group on the side chain, removal of theside-chain functional group, addition of a functional group (e.g.,hydroxyl group on proline), attachment of mono- or di-carbohydrate(e.g., via glycosylation) etc. Exemplary glycosylated derivativesinclude hydroxyl group on serine that is glycosylated with glucose,galactose, ribose, arabinose, xylose, lyxose, allose, altrose, mannose,gulose, iodose, talose, fucose, rhamnose, etc. as well as disaccharidesand amino sugars such as galactosamine, glucosamine, sialic acid,N-acetyl glucosamine, etc. Amino acid derivatives also include modifiedor unmodified D-amino acids.

The term “combinations thereof,” which reference to any modifications(e.g, carbohydrate modifications) of Ang-(1-7) derivatives refers tooligopeptides in which two, three, four, five, six, seven, or eight ofthe individual amino acids are modified by the attachment of acarbohydrate. For Ang-(1-7) derivatives having a plurality ofcarbohydrate modifications, the modifying carbohydrates may be the sameon every modified amino acid, or the several modified amino acids maycomprise a mixture of different carbohydrates.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal, at an appropriate interval and fora sufficient duration for treating a disease, is sufficient to effectsuch treatment for the disease. The “therapeutically effective amount”will vary depending on the compound, the disease and its severity,physiological factors unique to the individual including, but notlimited to the age, weight, and body mass index, the unitary dosage,cumulative dosage, frequency, duration, and route of administrationselected.

As used herein, the term “treating”, “contacting” or “reacting” whenreferring to chemical synthesis means to add or mix two or more reagentsunder appropriate conditions to produce the indicated and/or the desiredproduct. It should be appreciated that the reaction which produces theindicated and/or the desired product may not necessarily result directlyfrom the combination of two reagents which were initially added, i.e.,there may be one or more intermediates which are produced in the mixturewhich ultimately leads to the formation of the indicated and/or thedesired product.

As used herein, the terms “treating” and “treatment” refer to effectingan improvement of any symptom or physiological, cognitive, orbiochemical indicium of the condition or disease being treated. Forexample, treatment of a cognitive dysfunction and/or impairment mayrefer to: (1) preventing cognitive dysfunction and/or impairment fromoccurring, i.e., causing the clinical symptoms of cognitive dysfunctionand/or impairment not to develop in a subject that may be or predisposedto developing cognitive dysfunction and/or impairment but does not yetexperience or display symptoms of cognitive dysfunction and/orimpairment; (2) inhibiting cognitive dysfunction and/or impairment,i.e., arresting or reducing the development of cognitive dysfunctionand/or impairment or its clinical symptoms; or (3) relieving cognitivedysfunction and/or impairment, i.e., causing regression of cognitivedysfunction and/or impairment or its clinical symptoms.

The terms “approximately” or “about” in reference to a number aregenerally taken to include numbers that fall within a range of 5%, 10%,15%, or 20% in either direction (greater than or less than) of thenumber unless otherwise stated or otherwise evident from the context(except where such number would be less than 0% or exceed 100% of apossible value).

The term “subject” or “patient” refers to any organism to which acomposition of this invention may be administered, e.g., forexperimental, diagnostic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, dogs, cats,non-human primates, and humans).

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

Oligopeptides of the Invention: Some aspects of the invention provideoligopeptides that are derivatives of Ang-(1-7). As discussed above, theterm “derivative” of Ang-(1-7) refers to an oligopeptide whose aminoacid sequence of any one or more of Ang-(1-7) is modified (e.g., viamethylation, presence of a functional group, such as hydroxy group onproline), attached to a carbohydrate, is replaced with correspondingD-amino acid or an “equivalent amino acid” as defined above, and/or theterminal amino group end or the carboxyl end of Ang-(1-7) is modified,for example, the carboxylic acid end can be modified to be an amide, anamine, a thiol, or an alcohol functional group, or one in which anadditional amino acid residue is present compared to native Ang-(1-7).It should be appreciated that the term “Ang-(1-7) derivative” excludesthe native Ang-(1-7), i.e., amino acid sequences of endogenous Ang-(1-7)without any modification.

In some embodiments, oligopeptides of the invention have the amino groupon the carboxylic acid terminal end (i.e., the —OH group of thecarboxylic acid is replaced with —NR^(a)R^(b), where each of R^(a) andR^(b) is independently hydrogen or C,-C₆ alkyl) and/or have one or moreamino acid residues that are (i) replaced with a corresponding D-aminoacid, (ii) glycosylated, (iii) replaced with another amino acid, (iv) ora combination thereof.

Still in other embodiments, the oligopeptide of the invention isretro-inverso Ang-(1-7). Yet in other embodiments, the oligopeptide ofthe invention is retro Ang-(1-7). In other embodiments, the oligopeptideof the invention is inverso Ang-(1-7).

Other embodiments of the invention include Ang-(1-7) derivatives inwhich at least one or more, typically one or two, and often only oneamino acid is attached to a carbohydrate. Generally, the carbohydrate isattached to the amino acid via glycosylation. Typically, thecarbohydrate is a mono- or di-carbohydrate. Exemplary mono- anddi-carbohydrates that can be used in the invention include, but are notlimited to, xylose, fucose, rhamnose, glucose, lactose, cellobiose,melibiose, and a combination thereof.

In one particular embodiment, the oligopeptide of the invention isAng-(1-7) derivative of the formula: A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸ (SEQ IDNO:1), where A¹ is selected from the group consisting of aspartic acid,glutamic acid, alanine, and a derivative thereof A² is selected from thegroup consisting of arginine, histidine, lysine, and a derivativethereof A³ is selected from the group consisting of valine, alanine,isoleucine, leucine, and a derivative thereof A⁴ is selected from thegroup consisting of tyrosine, phenylalanine, tryptophan, and aderivative thereof A⁵ is selected from the group consisting ofisoleucine, valine, alanine, leucine, and a derivative thereof A⁶ isselected from the group consisting of histidine, arginine, lysine, and aderivative thereof A⁷ is selected from the group consisting of proline,glycine, serine, and a derivative thereof; and A⁸ can be present orabsent, wherein when A⁸ is present, A⁸ is selected from the groupconsisting of serine, threonine, hydroxyproline, and a derivativethereof, provided (i) at least one of A¹-A⁸ is optionally substitutedwith a mono- or di-carbohydrate; or (ii) when A⁸ is absent: (a) at leastone of A¹-A⁷ is substituted with a mono- or di-carbohydrate, (b) A⁷ isterminated with an amino group, or (c) a combination thereof.

In some embodiments, A¹ is the amino terminal end of the oligopeptideand A⁸ (or A⁷ when A⁸ is absent) is the carboxyl terminal end. Still inother embodiments, A¹ is the carboxyl terminal end and A⁸ (or A⁷ when A⁸is absent) is the amino terminal end. Yet in other embodiments, thecarboxylic acid functional group of the carboxyl terminal end ismodified as an amide functional group, an amine functional group, ahydroxyl functional group, or a thiol functional group. The amide andthe amine functional groups can be non-alkylate, mono-alkylated ordi-alkylated.

Yet in other embodiments, the carbohydrate comprises glucose, galactose,xylose, fucose, rhamnose, or a combination thereof. In some instances,the carbohydrate is a mono-carbohydrate, whereas in other instances, thecarbohydrate is a di-carbohydrate.

In other embodiments, at least one of A¹-A⁸ is substituted with amono-carbohydrate. Still in other embodiments, at least one of A¹-A⁸ issubstituted with a di-carbohydrate. It should be appreciated that thescope of the invention also includes those oligopeptides having bothmono- and di-carbohydrates.

Exemplary di-carbohydrates that can be used in oligopeptides of theinvention include, but are not limited to, lactose, cellobiose,melibiose, and a combination thereof. However, it should be appreciatedthat the scope of the invention includes oligopeptides that aresubstituted with any dicarbohydrates known to one skilled in the art.

In one particular embodiment, A⁸ is serine or a derivative thereof. Insome instances, the carboxylic acid moiety of the serine is modified asan amide or an amine. In one case, serine is terminated as an aminogroup. Still in other embodiments, the serine residue of A⁸ isglycosylated with glucose or lactose.

Yet in other embodiments, at least one, typically at least two,generally at least three, often at least four, still more often at leastfive, yet still more often at least six, and most often all of A¹-A⁸ isD-amino acid.

In particular, in some specific embodiments, said oligopeptide is retromodified, inverso modified, or retro-inverso modified.

Another aspect of the invention provides oligopeptides, such asAng-(1-7) derivatives, having eight amino acids or less, typically sevenor eight amino acid residues. In some embodiments, one or more aminoacids have attached thereto a carbohydrate group. Often the carbohydrategroup is attached to the oligopeptide via glycosylation. Thecarbohydrate can be attached to the oligopeptide via any of the sidechain functional group of the amino acid or the amide group.Accordingly, the scope of the invention includes, but is not limited to,O-glycosylate, N-glycosylate, S-glycosylated oligopeptides. The term“X-glycosylated” refers to having a carbohydrate attached to theoligopeptide via the heteroatom “X” of the amino acid. For example, forserine whose side-chain functional group is hydroxyl, “O-glycosylated”means the carbohydrate is attached to the serine's side-chain functionalgroup, i.e., the hydroxyl group. Similarly, “N-glycosylation” of leucinerefers to having the carbohydrate attached to the amino side-chainfunctional group of leucine. Typically, the glycosylation is on theside-chain functional group of the amino acid.

In some embodiments, the Ang-(1-7) derivative is glycosylated withxylose, fucose, rhamnose, glucose, lactose, cellobiose, melibiose, or acombination thereof.

Yet in other embodiments, the carboxylic acid terminal end of saidglycosylated Ang-(1-7) derivative is substituted with an amino group.When referring to the carboxyl acid terminal end being substituted withan amino group, it means —OH group of the carboxylic acid is replacedwith —NH₂ group. Thus, the actual terminal end functional group is anamide, i.e., rather than having the oligopeptide being terminated at thecarboxylic acid terminal end with a functional group —CO₂H, thecarboxylic acid terminal end is terminated with an amide group (i.e.,—CO₂NR′₂, where each R′ is independently hydrogen or C₁-C₁₂ alkyl).Still in other embodiments, the carboxylic acid terminal group isterminated with a hydroxyl or a thiol group. In some embodiments, themodified carboxylic acid terminal group is used to attach thecarbohydrate, e.g., via glycosylation.

One of the purposes of the invention was to produce Ang-(1-7)derivatives to enhance efficacy of action, in vivo stabilization, and/orpenetration of the blood-brain barrier. Improved penetration of theblood-brain barrier facilitates cerebral entry of the Ang-(1-7)derivative of the invention, and, consequently, Mas activation, orintrinsic-efficacy. To improve (i.e., increase) penetration of theblood-brain barrier, in some embodiments the Ang-(1-7) derivative isattached to at least one mono- or di-carbohydrates.

Without being bound by any theory, it is believed that the oligopeptideof the invention that are glycosylated exploits the inherentamphipathicity of the folded Ang-(1-7) glycopeptides (i.e., glycosylatedoligopeptides of the invention) and the “biousian approach” to deliverthe glycosylated oligopeptides of the invention across the blood-brainbarrier. In some instances, the amount of increase in crossing theblood-brain barrier by oligopeptides of the invention is at least 6%,typically at least 10%, and often at least 15% compared to nativeAng-(1-7). In other instances, oligopeptides of the invention have invivo half-life of at least 30 min, typically at least 40 min, and oftenat least 50 min. Alternatively, compared to native Ang-(1-7),oligopeptides of the invention exhibit at least 50 fold, typically atleast 75 fold, and often at least 100 fold increase in in vivohalf-life.

In other embodiments, oligopeptides of the invention exhibit enhancedvascular efficacy. Without being bound by any theory, it is generallyrecognized that blood-brain barrier transport occurs via an absorptiveendocytosis process on the blood side of the endothelium of the braincapillaries followed by exocytosis on the brain side, leading to overalltranscytosis. It is also believed that for this process to be efficient,the oligopeptide must bind to the membrane for some period of time, andmust also be able to exist in the aqueous state for some period of time(biousian nature). Based on previous work from one of the presentinventors, it is believed that effective drug delivery and blood-brainbarrier transport requires a biousian glycopeptide that has at least twostates: (1) a state defined by one or more membrane-bound conformationsthat permit or promote endocytosis; and (2) a state defined by awater-soluble, or random coil state that permits “membrane hopping” and,presumably, vascular efficacy.

In general, the degree of glycosylation does not have a large effect onthe structure of the individual microstates. Thus, altering the degreeof glycosylation allows for the modulation of aqueous vs. membrane-boundstate population densities without significantly affecting the overallstructure of the oligopeptide. Moreover, it is believed thatglycosylation also promotes stability to peptidases, thereby increasingthe half-life of the Ang-(1-7) derivatives in vivo.

TABLE 1 Some of the representative oligopeptides of the invention.carboxyl terminal end 1 2 3 4 5 6 7 8 functional group Native AT₁₋₇ AspArg Val Tyr Ile His Pro — OH (SEQ ID NO: 2) PN-A1 Asp Arg Val Tyr IleHis Pro — NH₂ (SEQ ID NO: 6) PN-A2 Asp Arg Val Tyr Ile His Pro Ser° NH₂(SEQ ID NO: 7) PN-A3 Asp Arg Val Tyr Ile His Pro Ser* NH₂ (SEQ ID NO: 8)PN-A4 Asp Arg Val Tyr Ile His Pro Ser** NH₂ (SEQ ID NO: 9) PN-A5 Asp ArgVal Tyr Ile His Ser* — NH₂ (SEQ ID NO: 10) PN-A6-PN-A11 Ala → scan TyrIle etc. Pro Ser°^(\)*^(/)** NH₂ (SEQ ID NO: 11) PN-A12 Asp Arg Xxx TyrYyy His Pro Ser°^(\)*^(/)** NH₂ (SEQ ID NO: 12) PN-AXX Asp Arg Xxx ZzzYyy His Pro Ser°^(\)*^(/)** NH₂ (SEQ ID NO: 13)

Table 1 above shows some of the representative oligopeptides of theinvention. In particular, these oligopeptides can be consideredAng-(1-7) derivatives. In Table 1, the “n-x”, where x is an integer,represents the oligonucleotide identifier. For example, PN-A1 meansoligopeptide number 1, PN-A2 means oligopeptide number 2, PN-A6-N-A11means oligopeptide numbers 6 through 11, and so forth. Thus, the term“A-x” is used for identification purposes only. As shown in Table 1,some of the oligopeptides have carbohydrate attached to the nativeAng-(1-7) peptide. These peptides are sometimes referred to asglycopeptides.

Studies have shown that inherent binding of the glycopeptide to thenative receptor is minimally affected. Therefore, the glycosylatedAng-(1-7) derivatives, at a minimum, maintain Mas binding similar tothat of the native Ang-(1-7) peptide. In addition, promoting the aqueousnature of the glycopeptide can further enhance vascular efficacy ofAng-(1-7) derivatives. The degree of glycosylation (e.g., Table 1:unglycosylated Ser°, glucosylated Ser* or lactosylated Ser**) foroptimal blood-brain barrier transport is determined using the bestbinding compounds from these using the in vivo mouse model. Besides thedisaccharide β-lactose, the more robust disaccharide β-cellobiose isexamined using these first few structures. Based on the amino acidsequence of Ang-(1-7) and the potential modification strategies, thereare at least about 200 possible derivatives of Ang-(1-7) that arerapidly generated using the well known oligopeptide synthesis, includingautomated peptide synthesis as well as combinatorial synthesis.

Other aspects of the invention provide methods for treating cognitivedysfunction and/or impairment in a patient using an oligopeptide of theinvention. Typically, methods of the invention include administering toa patient in need of such a treatment a therapeutically effective amountof an oligopeptide of the invention. It should be appreciated that theoligopeptides of the invention can be used to treat any clinicalconditions that are known to be treatable or appears to be treatableusing Ang-(1-7). However, for the sake or clarity and brevity, theinvention will now be described in reference to treating cognitivedysfunction and/or impairment in a patient.

The cognitive dysfunction that occurs in congestive heart failure (CHF)patients includes decreased attention, memory loss, psychomotor slowing,and diminished executive function, all of which compromises patients'ability to comply with complex medical regimens, adhere to dietaryrestrictions and make self-care decisions. Mechanisms thought tocontribute to cognitive impairment in patients with CHF include changesin cerebral blood flow, altered cerebrovascular autoregulation andmicroembolisms. In one study, cerebral blood flow was measured withsingle-photon emission computed tomography (SPECT) and found to bereduced by 30% in patients with severe heart failure. The causes fordecreased cerebral perfusion in CHF have been attributed to low cardiacoutput, low blood pressure and altered cerebrovascular reactivity. Insome cases, the cognitive impairment seen in CHF is improved followingeither heart transplant or improvement in cerebral blood flow viaoptimal management of CHF. However, for many patients with CHF,management is rarely optimal and the cognitive impairment persists.Interestingly, long-term follow up studies have revealed thatcognitively normal CHF patients have a significantly higher risk ofdementia or Alzheimer's disease compared to age-matched non-CHFcontrols, suggesting that CHF and cardiovascular disease predisposepatients to further cognitive impairment and dementia.

During CHF, the well characterized changes in the circulatingneurochemical milieu and increases in inflammatory factors are also seenin the brain. Most of the studies on CHF-induced changes in inflammatorycytokines and ROS have focused on brain regions involved in sympatheticoutflow regulation and not on cognition. CHF elevates sympathetic toneand causes abnormal cardiac and sympathetic reflex function. In the rat,ischemia-induced CHF significantly increases pro-inflammatory cytokinesand Angiotensin II type 1 receptors (AT1) in the paraventricular nucleus(PVN) of the hypothalamus. Further, in CHF rabbits, the increase insympathetic outflow is blocked by ICV injection of the super oxidedimustase (SOD) mimetic tempol, presumably by inhibition of ROS. CHF inthis model is associated with increased expression of NADPH oxidasesubunits and ROS production in the rostral ventral lateral medulla(RVLM) and increases in NO.

The role of ROS in learning and memory has been extensively studied. Allof the NAD(P)H oxidase subunits, including NOX2 and NOX4, have beenlocalized within the cell bodies and dendrites of neurons of the mousehippocampus and perirhinal cortex and are co-localized at synapticsites. These are key regions of the brain in learning and memory. In thebrain, superoxide production via actions of NAD(P)H oxidase are known tobe involved in neurotoxicity, age related dementia, stroke andneurodegenerative diseases and have been identified throughout the brainincluding the hippocampus, thalamus, cerebellum and amygdala. Inyounger, healthy animals ROS and NAD(P)H oxidase is shown to be requiredfor normal learning and hippocampal long-term potentiation (LTP). Recentstudies in mice lacking Mas have shown that Ang-(1-7) and Mas areessential for normal object recognition processing and blockade of Masin the hippocampus impairs object recognition. In addition, Ang-(1-7)facilitates LTP in CA1 cells and this effect is blocked by antagonism ofMas. In older animals or in CHF animals, an increase in ROS is linked toLTP and memory impairments.

Over the last decade, it has become recognized that renin angiotensinsystem (RAS) involves two separate enzymatic pathways providing aphysiological counterbalance of two related peptides acting at distinctreceptors. The well described ACE-AngII-AT1 receptor system is thoughtto be physiologically opposed and balanced by the ACE2-Ang-(1-7)-Massystem. Functionally, these two separate enzymatic pathways of RAS arethought to be involved in balancing ROS production and nitric oxide (NO)in the brain, microvasculature and peripheral tissues. Increases in AT1receptor activation are known to increase NAD(P)H oxidase and ROSgeneration which are both known to contribute to abnormal increases ofsympathetic nerve activity observed in CHF and hypertension. Thisincrease in AT1 receptor-induced ROS formation is thought to be opposedby ACE2-Ang-(1-7)-Mas inhibition of ROS formation. Ang-(1-7), themajority of which is produced from ACE2 cleavage of Ang II, decreasesROS production and increases NOS in the brain via activation Mas and,possibly through AT2 receptor.

Within the brain, the Mas receptor is known to be expressed on neurons,microglia and vascular endothelial cells. Further, all three of thesekey components that make up the “neurovascular unit” (neurons, microgliaand endothelial cells) are central players in neurogenic hypertensionand CHF-induced increases in brain inflammation and ROS production. BothCHF and hypertension increase circulating cytokines promoting ROSproduction within the “neurovascular unit”. The end-result of thisfeed-forward cascade is neuronal dysfunction and cognitive impairment.The ideal therapeutic candidate to treat cognitive impairment would bedesigned to interrupt this cascade by working at both sides of theblood-brain barrier, the brain vascular endothelium and neuronal cells.Ang-(1-7), acting at the Mas receptor, is known to have effects at bothendothelial cells and neurons. However, using a native Ang-(1-7) fortreating cognitive dysfunction and/or impairment is not suitable becausenative Ang-(1-7) is susceptible to enzymatic degradation. Moreover,native Ang-(1-7) does not readily cross the blood-brain barrier to besuitable as a therapeutic agent.

Without being bound by any theory, it is believed that one of theadvantages of using oligopeptides of the invention in treating cognitivedysfunction and/or impairment is that oligopeptides of the inventionhave enhanced endothelial “interaction” and brain penetration. It isbelieved that oligopeptides of the invention act at both endothelialcells and neurons thus inhibiting inter alia neurovascular ROSproduction and mitigating the brain inflammatory cascade.

Accordingly, oligopeptides the invention can be used to treat cognitiveimpairment and/or dysfunction (1) associated with pre- and/orpost-surgery dementia, or (2) observed in patients with congestive heartfailure, cardiovascular disease, or hypertension. More generally,oligopeptides of the invention are useful in treating cognitivedysfunction and/or impairment in a subject whose cognitive dysfunctionand/or impairment is clinically associated with an increase ininflammatory cytokines and/or increase in reactive oxygen species(“ROS”) in the central nervous system, in particular the brain. As usedherein, the term “clinically associated” refers to the root cause orunderlying cause of cognitive dysfunction and/or impairment (such as,but not limited to, memory loss) that when ameliorated results inreduction, prevention, treatment or reversal of cognitive dysfunctionand/or impairment. Exemplary clinical conditions associated with anincrease in inflammatory cytokines and/or increase in reactive oxygenspecies that can cause cognitive dysfunction and/or impairment include,but are not limited to, circulatory compromise, cardiovascular disease,hypertension, hypotension, congestive heart failure, stroke, embolism,surgery (e.g., postoperative recovery condition), dementia, Alzheimer'sdisease, disease related cognitive impairment, trauma related cognitiveimpairment, age-related dementia, postoperative related delirium and/orincrease in inflammatory cytokine and/or increase in reactive oxygenspecies within the central nervous system of said subject or acombination thereof.

Oligopeptides of the present invention can be administered to a patientto achieve a desired physiological effect. Preferably the patient is ananimal, more preferably a mammal, and most preferably a human. Theoligopeptide can be administered in a variety of forms adapted to thechosen route of administration, i.e., orally or parenterally. Parenteraladministration in this respect includes administration by the followingroutes: intravenous; intramuscular; subcutaneous; intraocular;intrasynovial; transepithelially including transdermal, ophthalmic,sublingual and buccal; topically including ophthalmic, dermal, ocular,rectal and nasal inhalation via insufflation and aerosol;intraperitoneal; and rectal systemic.

The active oligopeptide can be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it can beenclosed in hard or soft shell gelatin capsules, or it can be compressedinto tablets. For oral therapeutic administration, the activeoligopeptide may be incorporated with excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparation can contain at least 0.1% of active oligopeptide. Thepercentage of the compositions and preparation can, of course, be variedand can conveniently be between about 1 to about 10% of the weight ofthe unit. The amount of active oligopeptide in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.Preferred compositions or preparations according to the presentinvention are prepared such that an oral dosage unit form contains fromabout 1 to about 1000 mg of active oligopeptide.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin can be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier. Various other materials can be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules can be coated with shellac,sugar or both. A syrup or elixir can contain the active oligopeptide,sucrose as a sweetening agent, methyl and propylparabens apreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active oligopeptide can be incorporated intosustained-release preparations and formulation.

The active oligopeptide can also be administered parenterally. Solutionsof the active oligopeptide can be prepared in water suitably mixed witha surfactant such as hydroxypropylcellulose. Dispersion can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacterial and fungi. Thecarrier can be a solvent of dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g., sugars or sodium chloride. Prolonged absorption of theinjectable compositions of agents delaying absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activeoligopeptide in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

The therapeutic oligopeptides of the present invention can beadministered to a mammal alone or in combination with pharmaceuticallyacceptable carriers, as noted above, the proportion of which isdetermined by the solubility and chemical nature of the oligopeptide,chosen route of administration and standard pharmaceutical practice.

The physician will determine the dosage of the present therapeuticagents which will be most suitable for prophylaxis or treatment and itwill vary with the form of administration and the particularoligopeptide chosen, and also, it will vary with the particular patientunder treatment. The physician will generally wish to initiate treatmentwith small dosages by small increments until the optimum effect underthe circumstances is reached. The therapeutic dosage can generally befrom about 0.1 to about 1000 mg/day, and preferably from about 10 toabout 100 mg/day, or from about 0.1 to about 50 mg/Kg of body weight perday and preferably from about 0.1 to about 20 mg/Kg of body weight perday and can be administered in several different dosage units. Higherdosages, on the order of about 2× to about 4×, may be required for oraladministration.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

Ang-(1-7) derivative high-throughput screening (HTS): For HTS, asensitive and direct measure of nitric oxide (NO) production in 2separate cell lines is utilized, primary CA1 hippocampal neurons andhuman umbilical vein endothelial cells (HUVEC). The use of primary CA1cells is self-evident for the study of central effects. In addition, thecontribution of endothelial dysfunction to the progression of CHF and tothe induction of cognitive impairment is clinically appreciated. Theemerging picture that the Ang-(1-7) singling axis holds promise as atherapeutic target for endothelial dysfunction strongly indicates thatreversal of CHF-induced endothelial dysfunction as mechanism cannot beruled out. HUVEC are isolated from the human umbilical vein andcryo-preserved after primary culture. HUVEC is included as a second linefor the primary screen because these cells are the model in vitro systemfor the study of endothelial cell function and can be used to directlymeasure Mas-dependent NO production.

Cell culture. To isolate primary hippocampal CA1 neuronal cells, wholebrain was removed from neonatal rat pups (1-2 day old) and the corticesdissected away. The hippocampus was isolated and the CA1 field wasexcised and placed in buffer. After gentle disruption in digestionbuffer, the cells were counted, placed in culture media, and plated in a96-well format coated with poly-d-lysine. At the time of plating, cellswere approximately at 50% density and were allowed to culture to 70-80%density before starting the assay. Commercially available HUVEC (LifeTechnologies/Thermo Fisher) was thawed and plated (5000-10,000cells/well) in a 96-well format coated with gelatin. HUVEC cells wereallowed to culture overnight before starting the assay.

Cell Activation: The xCELLigence system Real-Time Cell Analyzer (RTCA),developed by Roche Applied Science, uses microelectronic biosensortechnology to do dynamic, real-time, label-free, and non-invasiveanalysis of cellular events including G-protein receptor activation ofcells. The RTCA analysis was utilized to measure the potency andrelative ability of oligopeptides of the invention and native Ang-(1-7)to activate human umbilical vascular endothelial cells (HUVEC) inculture. Following uniform cellular adherence based on a linear increasein cell impedance (CI), HUVECs were treated with Ang-(1-7) andoligopeptides of the invention. Each trace of the CI over time in FIG. 1represents the average of 4 wells normalized to CI at the time ofcompound addition. FIG. 1 shows the results from data acquired using thexCELLigence RTCA to measure the relative potency of PN-A3, PN-A4, PN-A5and native Ang-(1-7). A 100 nM administration of PN-A3, PN-A4 and PN-A5and 10 nM of PN-A3 and PN-A5 resulted in a significant (˜2-fold)increase in CI over the native Ang-(1-7) demonstrating that theoligopeptides of the invention have greater potency for cell activationthan native Ang-(1-7).

NO production assay. As a screen for mechanisms of action ofoligopeptides of the invention, the ability to increase NO production ofthree oligopeptides of the invention (PN-A3, PN-A4 and PN-A5) werecharacterized and compared to native Ang-(1-7). Human umbilical vascularendothelial cells (HUVEC) culture plates received fluorescence reactionbuffer (0.2 M phosphate buffer, pH 7, 1 mM EDTA, 0.1% glucose)containing diaminofluorescein-FM diacetate (DAF-FM, 1 μM) to measurereal-time NO production. Time-resolved (10 minutes) fluorescentintensity was detected using a BioTek Synergy 2 microplate reader withexcitation at 485 nm and emission at 535 nm. DAF-FM is a sensitiveflourometric derivative for the selective detection of NO in live cells.

FIG. 2 shows relative peak fluorescence intensity following 5 minutesexposure to native Ang-(1-7) and three oligopeptides of the invention.Values were normalized to control fluorescence. As expected, nativeAng-(1-7) induced a significant elevation of NO over control levels.More importantly, as shown in FIG. 2, oligopeptides of the invention(namely PN-A3, PN-A4 and PN-A5) also elicited a significant elevation ofNO over control levels, with PN-A5 significantly enhancing NO productionover that seen with native Ang-(1-7), *=p<0.05. These resultsdemonstrate that oligopeptides of the invention increase NO productionsimilar to or greater than that of native Ang-(1-7).

FIG. 3A illustrates the ability of the select Mas receptor antagonists,A779, (C39H60N₁₂O₁₁) which is known to block native Ang-(1-7) NOproduction, to also block NO production induced by the oligopeptide ofthe invention, namely PN-A5. In these studies, HUVEC cells wereincubated with DAF-FM, 1 μM to measure real-time NO production. Cellswere treated with either PN-A5 alone (1.0 mM, n=10), PN-A5+A779 (n=6).Measurements were obtained using an Olympus 550 Confocal Microscope andanalyzed using Image J. Images were obtained every 10 sec. These resultsindicate that the oligopeptide PN-A5 actions are due to activation ofthe Mas receptor.

FIG. 3 b shows the averaged effect of the select Mas receptorantagonists, A779, which is known to block native Ang-(1-7) NOproduction, to also block NO production induced by the oligopeptide ofthe invention, PN-A5. In these studies, HUVEC cells were incubated withDAF-FM, 1 μM to measure real-time NO production. Cells were treated witheither PN-A5 alone (1.0 mM, n=10), PN-A5+A779 (n=6), or the NO donorS-nitroso-N-acetylpenicillamine (SNAP). Fluorescent measurements wereobtained using an Olympus 550 Confocal Microscope and analyzed usingImage J. Images were obtained every 10 sec. The NO response produced byPN-A5 was completely blocked by A779 demonstrating that PN-A5's abilityto increase NO is due to PN-A5 actions on the Mas receptor.

E ects o An -(1-7) Derivative on Heart Failure (HF) Induced CognitiveImpairment: A total of 33, male C57B1/6J adult mice (Harlan, 8-10 weeksold) were used. Mice were randomly assigned to either the sham (n=12) orcongestive heart failure (CHF) group (n=21). Experimental groups aredescribed as follows: sham+saline, CHF+saline, CHF+PN-A5. All mice priorto surgery were weighed and anesthetized. For the CHF mice, MI wasinduced by ligation of the left coronary artery (LCA). Under anesthesia(2.5% isoflurane in a mixture of air and O₂) a thoracotomy was performedat the fourth left intercostal space and the LCA permanently ligated toinduce a myocardial infarction (MI). Occlusion of the LCA was confirmedby observing blanching, a slight change in color of the anterior wall ofthe left ventricle downstream of the ligature. Sham mice underwent thesame procedure with the exception of ligating the LCA.

Following 8 weeks post MI surgery, CHF mice were treated with eitherdaily subcutaneous injections of the Ang-(1-7) derivative PN-A5 (1mg/kg/day) for 28 days or saline. After 21 days, animals were tested forobject recognition using a standard NOR test as described below. Afterapproximately 25 days of treatment, animals were tested for spatialmemory using the standard Morris water task as described below.

Novel Object Recognition (NOR): The apparatus consisted of an evenlyilluminated Plexiglas box (12 cm×12 cm×12 cm) placed on a table insidean isolated observation room. All walls of the apparatus were covered inblack plastic, and the floor was grey with a grid that was used toensure that the location of objects did not change between objectfamiliarization and test phases. The mouse behavior and exploration ofobjects was recorded with a digital camera. The digital image from thecamera was fed into a computer in the adjacent room. Two digitalstopwatches were used to track the time the mouse spent interacting withthe objects of the test. All data was downloaded to Excel files foranalysis. Triplicate sets of distinctly different objects were used forthe test.

The novel object recognition task included 3 phases: habituation phase,familiarization phase, and test phase. For the habituation phase, on thefirst and second day, mice were brought to the observation roomhabituated to the empty box for 10 min per day. On the third day, eachmouse had a “familiarization” trial with two identical objects followedby a predetermined delay period and then a “test” trial in which oneobject was identical to the one in the familiarization phase, and theother was novel. All stimuli were available in triplicate copies of eachother so that no object needed to be presented twice. Objects were madeof glass, plastic or wood that varied in shape, color, and size.Therefore, different sets of objects were texturally and visuallyunique. Each mouse was placed into the box the same way for each phase,facing the center of the wall opposite to the objects. To preclude theexistence of olfactory cues, the entire box and objects were alwaysthoroughly cleaned with 70% ethanol after each trial and between mice.During the familiarization phase, mice were allowed to explore the twoidentical objects for 4 min and then returned to their home cages. Aftera 2 hour delay, the “test phase” commenced. The mice were placed back tothe same box, where one of the two identical objects presented in thefamiliarization phase was switched to a novel one and the mouse wasallowed to explore these objects for another 4 min. Mouse “exploratorybehavior” was defined as the animal directing its nose toward the objectat a distance of ˜2 cm or less. Any other behavior, such as restingagainst the object, or rearing on the object was not considered to beexploration. Exploration was scored by an observer blind to the mouse'ssurgical group (CHF vs. Sham). Finally, the positions of the objects inthe test phases, and the objects used as novel or familiar, werecounterbalanced between the 2 groups of mice.

Discrimination ratios were calculated from the time spent exploring thenovel object minus time spent exploring the familiar object during thetest phase divided by the total exploration time. DRatio=(t novel−tfamiliar)/(t novel+t familiar). Data were analyzed from first 2 minutesof ‘test phase’. A positive score indicates more time spent with thenovel object, a negative score indicates more time spent with thefamiliar object, and a zero score indicates a null preference. All NORdata was examined using one-way analysis of variance, between subjects(ANOVA). Individual group differences were tested using the post hocTukey HSD test. In comparisons between groups of different sample sizes,equal variance was tested using a modified Levene's test. Allstatistical tests and p-values were calculated using MS Excel withDaniel's XLtoolbox and alpha was set at the 0.05 level. Error barsrepresent SEM.

Morris Water Task: Testing Spatial Learning and Memory/Visual Test: Theapparatus used was a large circular pool approximately 1.5 meters indiameter, containing water at 25° C. made opaque with addition ofnon-toxic white Crayola paint. An escape platform was hidden just belowthe surface of the water. Visual, high contrast cues were placed on thewalls of the test room. A digital camera connected to a computer in theadjacent room is suspended over the tank to record task progress. Forspatial testing prior to MI at 4 and 8 weeks post-MI or sham surgery,the platform was located at different sites in the pool.

During the spatial version of the Morris water task, all animals weregiven 6 training trials per day over 4 consecutive days. During thesetrials, an escape platform was hidden below the surface of water. Micewere released from seven different start locations around the perimeterof the tank, and each animal performed two successive trials before thenext mouse was tested. The order of the release locations waspseudo-randomized for each mouse such that no mouse was released fromthe same location on two consecutive trials. Performance on the swimtask was analyzed with a commercial software application (ANY-maze, WoodDale, IL). Because different release locations and differences inswimming velocity produce variability in the latency to reach the escapeplatform, a corrected integrated path length (CIPL) was calculated toensure comparability of mice performance across different releaselocations. The CIPL value measures the cumulative distance over timefrom the escape platform corrected by an animal's swimming velocity, andis equivalent to the cumulative search error. Therefore, regardless ofthe release location, if the mouse mostly swims towards the escapeplatform the CIPL value will be low. In contrast, the more time a mousespends swimming in directions away from the platform, the higher theCIPL value.

Following approximately 21 days of treatment with oligopeptide PN-A5,CHF mice showed object recognition memory improvement. FIG. 4illustrates the effects of three weeks treatment with oligopeptide PN-A5on object recognition memory as determined by the Novel ObjectRecognition Test (NOR). The mean performance of CHF mice witholigopeptide PN-A5 treatment (n=11) was similar to sham mice with saline(n=6), (CHF-Ang-(1-7) derivative PN-A5 M=+0.38, SE.11 vs. Sham-salineM=+0.52, SE 0.06) and significantly greater in comparison to CHF micetreated with saline (n=10) (M=−0.05, SE 0.09, *=p=0.009. These resultsdemonstrate that oligopeptide PN-A5 acts to attenuate and even rescueobject recognition memory impairment in mice with CHF.

Following approximately 25 days of treatment with oligopeptide PN-A5,CHF mice showed spatial memory improvement. FIG. 5 shows the mean CIPLof CHF+oligopeptide PN-A5 mice (n=11), CHF-saline treated mice (n=10)and Sham+saline mice (n=6). The CHF +oligopeptide PN-A5 mice showedsignificant improvement in spatial memory day 3 of the Morris swim taskas compared to CHF-saline mice. CHF mice treated with saline had asignificantly higher CIPL score as compared to CHF-oligopeptide PN-A5treated mice (CHF-saline M=32.5, SE=2.1 vs CHF-oligopeptide PN-A5M=23.5, SE 2.2, *=p=0.003. These results demonstrate that oligopeptidePN-A5 improves spatial memory.

Effect of oligopeptide PN-A5 on Nitric Oxide Bone Pain: FemaleBALB/cfC3H mice (Harlan, Ind, USA) were 15 to 20 g prior to initiationof study (n=5 animals per treatment group). Clinical signs of morbiditywere monitored and mice not meeting inclusion parameters (e.g.paralysis, rapid weight loss of >20% in 1 week) were removed from thestudy.

Mice were anesthetized with ketamine:xylazine (80 mg:12 mg/kg, 10 ml/kginjection volume; Sigma-Aldrich). An arthrotomy was performed. Thecondyles of the right distal femoris were exposed and a hole was drilledto create a space for injection of 4×10⁴ 66.1 cells in 5 μL Opti-MEM or5 μL Opti-MEM without cells in control animals within the intramedullaryspace of the mouse femoris. Injections were made with an injectioncannula affixed via plastic tubing to a 10-μL Hamilton syringe (CI31,Plastics One). Proper placement of the injector was confirmed throughuse of Faxitron X-ray imaging. Holes were sealed with bone cement.

Spontaneous pain (flinching and guarding), and tactile allodynia weremeasured 0, 15, 30, 60, 90 and 120 minutes after a single dose of drugwas administrated in a blinded fashion on Day 7. Breast cancer-inducedhypersensitivity returned to baseline levels 2 hours after drugadministration. Flinching and guarding were observed for duration of 2minutes during a resting state. Flinching was characterized by thelifting and rapid flexing of the right hind paw when not associated withwalking or movement. Flinches were recorded on a five-channel counter.Guarding was characterized by the lifting the right hind limb into afully retracted position under the torso. Time spent guarding over theduration of 2 minutes was recorded.

The assessment of tactile allodynia consisted of measuring thewithdrawal threshold of the paw ipsilateral to the site of tumorinoculation in response to probing with a series of calibrated von Freyfilaments using the Chaplan up-down method with the experimenter blindedto treatment groups. The 50% paw withdrawal threshold was determined bythe nonparametric method of Dixon.

On day 7, mice received an intraperitoneal (i.p.) injection of eithersaline or 0.8 g/μL (200 μL) for a total dose of 800 μg/kg. The in-vivoefficacy of PN-A5 was measured for a total of 2 hours.

Within group data were analyzed by non-parametric one-way analysis ofvariance. Differences were considered to be significant if P≦0.05. Alldata were plotted in GraphPad Prism 6.

FIG. 6 shows the results on the effects of oligopeptide PN-A5 on cancerinduced bone pain (CIBP). Cancer implanted into the distal femoralis ofmice induced a significant increase in the number of spontaneousflinches (FIG. 6A) and time spent guarding (FIG. 6B) after 7 days.Administration of a bolus of PN-A5 (800 μg/kg, i.p.) significantlyreversed cancer induces spontaneous pain for nearly one hour in duration(flinching: 60 min; guarding: 30 min; p<0.001). Similarly,cancer-induced tactile hypersenstivity was significantly attenuated 30minutes after injection (p<0.01). For all measurements, the time of peakeffect was 15-30 min. Behaviors returned to post-surgery values 90 minpost-injection. Media inoculated, sham control animals did were notstatistically different from pre-surgery baselines at any point duringtime-course.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. An oligopeptide derivative of the formula:A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸ (SEQ ID NO:1) wherein A¹ is selected from thegroup consisting of aspartic acid, glutamic acid, alanine, and aderivative thereof; A² is selected from the group consisting ofarginine, histidine, lysine, and a derivative thereof; A³ is selectedfrom the group consisting of valine, alanine, isoleucine, leucine, and aderivative thereof; A⁴ is selected from the group consisting oftyrosine, phenylalanine, tryptophan, and a derivative thereof; A⁵ isselected from the group consisting of isoleucine, valine, alanine,leucine, and a derivative thereof; A⁶ is selected from the groupconsisting of histidine, arginine, lysine, and a derivative thereof; A⁷is selected from the group consisting of proline, glycine, serine, and aderivative thereof; and A⁸ can be present or absent, wherein when A⁸ ispresent, A⁸ is selected from the group consisting of serine, threonine,hydroxyproline, and a derivative thereof, provided that when A⁸ isabsent: (a) at least one of A¹-A⁷ is substituted with a mono- ordi-carbohydrate, (b) A⁷ is terminated with an amino group, or (c) acombination thereof.
 2. The oligopeptide derivative of claim 1, whereinat least one of A¹-A⁷ is substituted with a mono- or di-carbohydrate. 3.The oligopeptide derivative of claim 1, wherein at least one of themono- or di-carbohydrates comprises glucose, galactose, xylose, fucose,rhamnose, lactose, cellobiose, melibiose.
 4. The oligopeptide derivativeof claim 1, wherein A⁸ is serine or a derivative thereof.
 5. Theoligopeptide derivative of claim 1, wherein (i) A⁸ is terminated with anamino group; or (ii) A⁸ is absent and A⁷ is terminated with an aminogroup.
 6. The oligopeptide derivative of claim 5, wherein (i) A⁸ isserine that is glycosylated with glucose or lactose; or (ii) A⁸ isabsent and A⁷ is serine that is glycosylated with glucose or lactose. 7.The oligopeptide derivative of claim 6, wherein A⁸ is absent and A⁷ isglycosylated with glucose.
 8. The oligopeptide derivative of claim 1,wherein A¹ is aspartic acid; A² is arginine; A³ is valine; A⁴ istyrosine; A⁵ is isoleucine; A⁶ is histidine; and (i) A⁸ is absent and A⁷either is terminated with an amino group or is a glycosylated serine, or(ii) A⁸ is serine terminated with an amino group.
 9. The oligopeptidederivative of claim 8, wherein A⁸ is a glycosylated serine.
 10. Theoligopeptide derivative of claim 8, wherein A⁸ is absent and A⁷ is aglycosylated serine that is terminated with an amino group.
 11. Theoligopeptide derivative of claim 1, wherein said oligopeptide derivativeis selected from the group consisting of SEQ ID NO:1; SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
 12. Aglycosylated Ang-(1-7) derivative having seven or eight amino acids. 13.The glycosylated Ang-(1-7) derivative of claim 12, wherein saidglycosylated Ang-(1-7) derivative is glycosylated with xylose, fucose,rhamnose, glucose, lactose, cellobiose, melibiose, or a combinationthereof.
 14. The glycosylated Ang-(1-7) derivative of claim 12, whereina carboxylic acid end of said glycosylated Ang-(1-7) derivative issubstituted with an amino group.
 15. A method for treating cognitiveimpairment in a patient, said method comprising administering to apatient in need of such a treatment a therapeutically effective amountof an oligopeptide of claim
 1. 16. A method for treating cognitiveimpairment in a patient, said method comprising administering to apatient in need of such a treatment a therapeutically effective amountof a glycosylated Ang-(1-7) derivative of claim 12.